Human immunodeficiency virus type 1 vpr induces apoptosis through caspase activation

Human immunodeficiency virus type 1 (HIV-1) Vpr is a 96-amino-acid protein that is found associated with the HIV-1 virion. Vpr induces cell cycle arrest at the G(2)/M phase of the cell cycle, and this arrest is followed by apoptosis. We examined the mechanism of Vpr-induced apoptosis and found that HIV-1 Vpr-induced apoptosis requires the activation of a number of cellular cysteinyl aspartate-specific proteases (caspases). We demonstrate that ectopic expression of anti-apoptotic viral proteins, which inhibit caspase activity, and addition of synthetic peptides, which represent caspase cleavage sites, can inhibit Vpr-induced apoptosis. Finally, inhibition of caspase activity and subsequent inhibition of apoptosis results in increased viral expression, suggesting that therapeutic strategies aimed at reducing Vpr-induced apoptosis in vivo require careful consideration.     

The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection.

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that can cause extensive cytopathicity in T cells. However, long-term productive infection of T-cell lines has been described. Here we show that although Vpr has no effect on the initial cytopathic effect of HIV-1, viruses that contain an intact vpr gene are unable to establish a chronic infection of T cells. However, virus with a mutated vpr gene can readily establish such long-term cultures. The effect of Vpr is independent of the env gene and the nef gene. Furthermore, expression of Vpr alone affects the progression of cells in the cell cycle. These results suggest that HIV-1 has evolved a viral gene to prevent chronic infection of T cells.     

Isolation of yeast mutants sensitive to the bifunctional alkylating agent nitrogen mustard

Summary Mutants of Saccharomyces cerevisiae with enhanced sensitivity to the DNA cross-linking agent nitrogen mustard (HN2) have been isolated and partially characterized with respect to their phenotypic and genetic properties. The screening technique, based on HN2-sensitivity as sole criterion, yields approximately 1 sensitive isolate in 200 clones when applied to an intensively mutagenized population of a resistant parent strain. Mutants characterized so far are all due to recessive nuclear genes and represent at least seven complementation groups. They exhibit different degrees as well as different patterns of sensitivity towards monofunctional and bifunctional alkylating agents, and ultraviolet light.     

Antisense phosphorothioate oligodeoxynucleotides targeted to the vpr gene inhibit human immunodeficiency virus type 1 replication in primary human macrophages.

The replication of human immunodeficiency viruses (HIV) in human macrophages is influenced by genetic determinants which have been mapped predominantly to the viral envelope. However, in HIV-2, the vpr gene has also been suggested as an important modulator of viral expression in human macrophages. We synthesized five antisense phosphorothioate oligodeoxynucleotides complementary to the vpr mRNA of HIV-1Ba-L, a highly macrophage-tropic viral strain, and measured their effect on HIV-1Ba-L replication in primary human macrophages. All of the oligodeoxynucleotides displayed some level of non-sequence-specific inhibition of viral replication; however, only the antisense one had an additional effect on viral production in primary macrophages. Of the five antisense oligodeoxynucleotides tested, only one did not show any additional effect on viral production, whereas all the others inhibited viral replication to a similar degree (70 to 100%). Variation in the degree of inhibition was observed by using five different donors of human primary macrophages. The phosphorothioate oligonucleotides, targeted to the initiating methionine of the Vpr protein, had an inhibitory effect at both 20 and 10 microM only when the size was increased from 24 to 27 bases. Thus, HIV-1 replication in human macrophages is modulated by the expression of the vpr gene, and it is conceivable that vpr antisense oligodeoxynucleotides could be used in combination with antisense oligodeoxynucleotides against other HIV-1 regulatory genes to better control viral expression in human macrophages.     

Human immunodeficiency virus type 1 Vpr induces DNA replication stress in vitro and in vivo

The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G2. Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4+ lymphocytes causes G2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4+ lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4+ lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24Gag-immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G2 arrest.     

Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest.

The human immunodeficiency virus type 1 (HIV-1) vpr gene encodes a protein which induces arrest of cells in the G2 phase of the cell cycle. Here, we demonstrate that following the arrest of cells in G2, Vpr induces apoptosis in human fibroblasts, T cells, and primary peripheral blood lymphocytes. Analysis of various mutations in the vpr gene revealed that the extent of Vpr-induced G2 arrest correlated with the levels of apoptosis. However, the alleviation of Vpr-induced G2 arrest by treatment with the drug pentoxifylline did not abrogate apoptosis. Together these studies indicate that induction of G2 arrest, but not necessarily continued arrest in G2, was required for Vpr-induced apoptosis to occur. Finally, Vpr-induced G2 arrest has previously been correlated with inactivation of the Cdc2 kinase. Some models of apoptosis have demonstrated a requirement for active Cdc2 kinase for apoptosis to occur. Here we show that accumulation of the hypophosphorylated or active form of the Cdc2 kinase is not required for Vpr-induced apoptosis. These studies indicate that Vpr is capable of inducing apoptosis, and we propose that both the initial arrest of cells and subsequent apoptosis may contribute to CD4 cell depletion in HIV-1 disease.     

Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme.

The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr.     

vpr deletion mutant of simian immunodeficiency virus induces AIDS in rhesus monkeys.

In previous experiments, animals infected with SIVmac239 containing a point mutation in the vpr and nef genes developed AIDS-like symptoms after early reversion of the vpr and nef genes. Here we show that two animals in which the nef gene but not the vpr gene had reverted in the first few months did not develop disease during a 3-year observation period even after reversion to a functional vpr gene 70 weeks postinfection. To study the influence of a stable vpr mutation on virus load and pathogenesis, a 43-bp deletion was introduced into the vpr gene of SIVmac239on, a nef-open mutant of SIVmac239. Four rhesus monkeys were inoculated with the vpr deletion mutant (SIV delta vpr), and two control animals were infected with SIVmac239on. Both control animals had persistent antigenemia, high cell-associated virus loads, and elevated neopterin levels. They had to be euthanized 20 and 30 weeks postinfection because of AIDS-related symptoms. However, all four rhesus monkeys inoculated with SIV delta vpr showed only transiently detectable antigenemia. The cell-associated virus loads were high in three of the four animals. Two animals with AIDS-like symptoms had to be euthanized 71 and 73 weeks postinfection. The two remaining monkeys infected with SIV delta vpr were still alive 105 weeks postinfection. In contrast to the SIVmac239on-infected animals, SIV delta vpr-infected animals had strong humoral immune responses and intermittent cellular immune responses to SIV antigens. Our data show that a functional vpr gene is not necessary for pathogenesis. However, vpr-deficient SIVmac239 variants might be slightly attenuated, allowing some animals to resist progression to disease for an extended period of time.     

Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4.

CD4 is an integral membrane glycoprotein which is known as the human immunodeficiency virus (HIV) receptor for infection of human cells. The protein is synthesized in the endoplasmic reticulum (ER) and subsequently transported to the cell surface via the Golgi complex. HIV infection of CD4+ cells leads to downmodulation of cell surface CD4, due at least in part to the formation of stable intracellular complexes between CD4 and the HIV type 1 (HIV-1) Env precursor polyprotein gp160. This process "traps" both proteins in the ER, leading to reduced surface expression of CD4 and reduced processing of gp160 to gp120 and gp41. We have recently demonstrated that the presence of the HIV-1-encoded integral membrane protein Vpu can reduce the formation of Env-CD4 complexes, resulting in increased gp160 processing and decreased CD4 stability. We have studied the effect of Vpu on CD4 stability and found that Vpu induces rapid degradation of CD4, reducing the half-life of CD4 from 6 h to 12 min. By using a CD4-binding mutant of gp160, we were able to show that this Vpu-induced degradation of CD4 requires retention of CD4 in the ER, which is normally accomplished through its binding to gp160. The involvement of gp160 in the induction of CD4 degradation is restricted to its function as a CD4 trap, since, in the absence of Env, an ER retention mutant of CD4, as well as wild-type CD4 in cultures treated with brefeldin A, a drug that blocks transport of proteins from the ER, is degraded in the presence of Vpu.     

Human immunodeficiency virus type 1 Vpr induces apoptosis in human neuronal cells

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) causes AIDS dementia complex (ADC) in certain infected individuals. Recent studies have suggested that patients with ADC have an increased incidence of neuronal apoptosis leading to neuronal dropout. Of note, a higher level of the HIV-1 accessory protein Vpr has been detected in the cerebrospinal fluid of AIDS patients with neurological disorders. Moreover, extracellular Vpr has been shown to form ion channels, leading to cell death of cultured rat hippocampal neurons. Based on these previous findings, we first investigated the apoptotic effects of the HIV-1 Vpr protein on the human neuronal precursor NT2 cell line at a range of concentrations. These studies demonstrated that apoptosis induced by both Vpr and the envelope glycoprotein, gp120, occurred in a dose-dependent manner compared to protein treatment with HIV-1 integrase, maltose binding protein (MBP), and MBP-Vpr in the undifferentiated NT2 cells. For mature, differentiated neurons, apoptosis was also induced in a dose-dependent manner by both Vpr and gp120 at concentrations ranging from 1 to 100 ng/ml, as demonstrated by both the terminal deoxynucleotidyltransferase (Tdt)-mediated dUTP-biotin nick end labeling and Annexin V assays for apoptotic cell death. In order to clarify the intracellular pathways and molecular mechanisms involved in Vpr- and gp120-induced apoptosis in the NT2 cell line and differentiated mature human neurons, we then examined the cellular lysates for caspase-8 activity in these studies. Vpr and gp120 treatments exhibited a potent increase in activation of caspase-8 in both mature neurons and undifferentiated NT2 cells. This suggests that Vpr may be exerting selective cytotoxicity in a neuronal precursor cell line and in mature human neurons through the activation of caspase-8. These data represent a characterization of Vpr-induced apoptosis in human neuronal cells, and suggest that extracellular Vpr, along with other lentiviral proteins, may increase neuronal apoptosis in the CNS. Also, identification of the intracellular activation of caspase-8 in Vpr-induced apoptosis of human neuronal cells may lead to therapeutic approaches which can be used to combat HIV-1-induced neuronal apoptosis in AIDS patients with ADC.     

Human immunodeficiency virus type 1 Nef induces accumulation of CD4 in early endosomes.

We have studied the fate of CD4 in CEM T cells expressing a human immunodeficiency virus type 1 HIV-1 Nef protein. Nef triggered a rapid endocytosis and a degradation of CD4, while most of the p56lck was upheld at the cell membrane. In the presence of Nef, CD4 accumulated in acidic intracellular vesicles that were not stained by antibodies against rab6, a marker of the Golgi apparatus complex. Detection of transferrin in CD4-containing vesicles showed that CD4 was trapped in early endosomes, without significant accumulation of CD4 in late endocytic compartments. Internalization pathways taken by CD4 in Nef+ cells may therefore be different from those observed after treatment with phorbol esters.     

Human immunodeficiency virus type 1 induces 1-beta-D-arabinofuranosylcytosine resistance in human H9 cell line.

We have found that chronically HIV-1(IIIB)-infected H9 cells showed 21-fold resistance to 1-beta-D-arabinofuranosylcytosine (ARA-C) compared with uninfected H9 cells. In the infected H9 cells, a 37% increase of dCTP pool and a 34% increase of dATP were observed, and no alteration of dTTP and dGTP was observed, compared with the uninfected H9 cells. A marked decrease of ARA-CTP generation was observed in the infected H9 cells after 3-h incubation with 0.1-10 microM ARA-C. The level of deoxycytidine kinase activity with ARA-C as substrate was similar in both the infected and the uninfected cells; however, a 37-fold increase of cytidine deaminase activity was observed in the infected H9 cells. These results indicate that the induction of cytidine deaminase activity by HIV-1(IIIB) infection conferred ARA-C resistance to H9 cells. This conclusion was supported by the observation that a marked reversal of ARA-C resistance in the infected H9 cells occurred after treatment with the inhibitor of cytidine deaminase, 3,4,5,6-tetrahydrouridine. The understanding of these cellular alterations in drug sensitivity may facilitate the development of effective therapeutic strategies against HIV-1-infected cells.     

Human immunodeficiency virus type 1 induces expression of complement factors in human astrocytes

Since the brain is separated from the blood immune system by a tight barrier, the brain-resident complement system may represent a central player in the immune defense of this compartment against human immunodeficiency virus (HIV). Chronic complement activation, however, may participate in HIV-associated neurodegeneration. Since the level of complement factors in the cerebrospinal fluid is known to be elevated in AIDS-associated neurological disorders, we evaluated the effect of HIV type 1 (HIV-1) on the complement synthesis of brain astrocytes. Incubation of different astrocytic cell lines and primary astrocytes with HIV-1 induced a marked upregulation of the expression of the complement factors C2 and C3. The synthesis of other secreted or membrane-bound complement proteins was not found to be altered. The enhancement of C3 production was measured both on the mRNA level and as secreted protein in the culture supernatants. HIV-1 laboratory strains as well as primary isolates were capable of inducing C3 production with varied effectiveness. The usage of viral coreceptors by HIV-1 was proved to be a prerequisite for the upregulation of C3 synthesis, which was modulated by the simultaneous addition of cytokines. The C3 protein which is secreted after incubation of the cells with HIV was shown to be biologically active as it can participate in the complement cascade.     

Comparison of cell cycle arrest, transactivation, and apoptosis induced by the simian immunodeficiency virus SIVagm and human immunodeficiency virus type 1 vpr genes

All primate lentiviruses known to date contain one or two open reading frames with homology to the human immunodeficiency virus type 1 (HIV-1) vpr gene. HIV-1 vpr encodes a 96-amino-acid protein with multiple functions in the viral life cycle. These functions include modulation of the viral replication kinetics, transactivation of the long terminal repeat, participation in the nuclear import of preintegration complexes, induction of G2 arrest, and induction of apoptosis. The simian immunodeficiency virus (SIV) that infects African green monkeys (SIVagm) contains a vpr homologue, which encodes a 118-amino-acid protein. SIVagm vpr is structurally and functionally related to HIV-1 vpr. The present study focuses on how three specific functions (transactivation, induction of G2 arrest, and induction of apoptosis) are related to one another at a functional level, for HIV-1 and SIVagm vpr. While our study supports previous reports demonstrating a causal relationship between induction of G2 arrest and transactivation for HIV-1 vpr, we demonstrate that the same is not true for SIVagm vpr. Transactivation by SIVagm vpr is independent of cell cycle perturbation. In addition, we show that induction of G2 arrest is necessary for the induction of apoptosis by HIV-1 vpr but that the induction of apoptosis by SIVagm vpr is cell cycle independent. Finally, while SIVagm vpr retains its transactivation function in human cells, it is unable to induce G2 arrest or apoptosis in such cells, suggesting that the cytopathic effects of SIVagm vpr are species specific. Taken together, our results suggest that while the multiple functions of vpr are conserved between HIV-1 and SIVagm, the mechanisms leading to the execution of such functions are divergent.     

Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr.

We investigated the fate of human immunodeficiency virus type 1 (HIV-1) viral DNA in infected peripheral blood lymphocytes and immortalized T-cell lines by using a replication-defective HIV-1. We observed that integrated HIV-1 DNA and viral gene expression decrease over time. A frameshift mutation in vpr resulted in maintenance of the HIV-1 provirus and stable persistence of viral expression. Transfection of vpr together with the neomycin resistance gene in the absence of other viral genes decreased the formation of geneticin-resistant colonies, indicating either a cytotoxic or a cytostatic effect upon cells. Therefore, maintenance of HIV-1 infection within an infected proliferating population is due to two competing processes, the rate of viral spread and the degree of cell growth inhibition and/or death induced by Vpr.